Proper control of the synthesis, storage and secretion of bioactive peptides is crucial to normal endocrine and neural function. Over the past several decades, we delineated the cell-type specific enzymatic steps leading from proopiomelanocortin (POMC) to its many product peptides. We selected one of these enzymes, peptidylglycine 1-amidating monooxygenase (PAM), as a focus of our studies because it is an integral membrane protein that can communicate information about the lumen of the regulated secretory pathway to cytosolic machinery and to the nucleus. Our focus on PAM led to the discovery of Kalirin, a GDP/GTP exchange factor (GEF) for small GTP binding proteins of the Rho family, that interacts with the cytosolic domain of PAM. Linkage studies associating Kalirin with coronary artery disease, the decrease in Kalirin expression associated with elevated iNOS in Alzheimer disease hippocampus, and the identification of Kalirin as one of the proteins essential for Ras-mediated epigenetic silencing of gene expression, motivate our studies of this complex, multidomain protein. By flanking an exon common to the major splice variants of Kalirin with loxp sites, Kalirin conditional knockout mice (KalCKO/CKO) and mice lacking the major isoforms of Kalirin (KalKO/KO) were created. While not yet well characterized, it is clear that normal storage and secretion of pituitary hormones require pituitary Kalirin. Using lentiviruses or mating with mice in which expression of Cre recombinase is driven by the POMC or growth hormone promoter, Kalirin expression in corticotropes or somatotropes will be eliminated. POMC and GH synthesis, processing and secretion will be evaluated in vivo and in cell culture. The hypothesis that G1q-mediated activation of the second GEF domain of Kalirin plays a key role in the ability of corticotropes to respond to specific secretagogues will be tested in vitro and in vivo. Endocytic trafficking of PAM will be evaluated to determine the role of Kalirin in recycling granule membrane proteins and PAM-mediated nuclear signaling. Proteomic analysis will be carried out on isolated immature granules, whose formation is regulated by Kalirin, and on isolated pituitary granules formed in the absence of Kalirin. Assays to detect activation of Rho GEFs and their effectors will be used to understand their physiological role in pituitary hormone secretion. Finally, we will use knowledge of its individual domains to test the hypothesis that Kalirin functions as a modular machine, coordinating multiple aspects of granule biogenesis and release. In particular, the physiological importance of the alternate N-termini of Kalirin, the ability of Kalirin to inhibit iNOS, and the ability of Kalirin to integrate signals from multiple pathways will be assessed in vivo and explored mechanistically in cell culture. PUBLIC HEALTH RELEVANCE: Precise control of the biosynthesis, storage and secretion of bioactive peptides requires the coordinate control of many different cellular processes. Genetically engineered mice were used to determine that Kalirin, a large protein with domains that allow it to interact with immature secretory granules, regulate the actin cytoskeleton, bind to lipid membranes and respond to multiple protein/protein interactions, plays an essential role in peptide hormone release. Mutations in the Kalirin gene or changes in Kalirin expression have been correlated with early-onset coronary artery disease, schizophrenia and Alzheimer Disease, making a better understanding of the functions of this complex protein relevant to human health.